BMC Pregnancy and Childbirth
BioMed Central
Open Access
Study protocol
Multiplex ligation-dependent probe amplification versus
karyotyping in prenatal diagnosis: the M.A.K.E. study
Elisabeth MA Boormans*1,2, Erwin Birnie3, Hajo I Wildschut4,
Heleen G Schuring-Blom5, Dick Oepkes6, Carla AC van Oppen7,
Jan G Nijhuis8, Merryn VE Macville9, Angelique JA Kooper10,
Karin Huijsdens11, Mariëtte VJ Hoffer12, Attie Go13, Johan Creemers14,
Shama L Bhola15, Katia M Bilardo1, Ron Suijkerbuijk16, Katelijne Bouman16,
Robert-Jan H Galjaard17, Gouke J Bonsel3 and Jan MM van Lith2,6
Address: 1Department of Obstetrics and Gynecology, Academic Medical Centre, Amsterdam, The Netherlands, 2Department of Obstetrics and
Gynecology, Onze Lieve Vrouwe Gasthuis, Amsterdam, The Netherlands, 3Institute of Health Policy and Management, Erasmus Medical Center,
Rotterdam, The Netherlands, 4Department of Obstetrics and Gynecology, Erasmus Medical Center, Rotterdam, The Netherlands, 5Department of
Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands, 6Department of Obstetrics and Gynecology, Leiden University
Medical Center, Leiden, The Netherlands, 7Department of Obstetrics and Gynecology, University Medical Center Utrecht, Utrecht, The
Netherlands, 8Department of Obstetrics and Gynecology, Academic Medical Center Maastricht, Maastricht, The Netherlands, 9Department of
Clinical Genetics, Academic Medical Center Maastricht, Maastricht, The Netherlands, 10Department of Human Genetics, Radboud University
Nijmegen Medical Center, Nijmegen, The Netherlands, 11Department of Clinical Genetics, Academic Medical Center, Amsterdam, The
Netherlands, 12Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands, 13Department of Obstetrics and
Gynecology, VU Medical Center, Amsterdam, The Netherlands, 14Department of Obstetrics and Gynecology, Radboud University Nijmegen
Medical Center, Nijmegen, The Netherlands, 15Department of Clinical Genetics, VU Medical Center, Amsterdam, The Netherlands, 16Department
of Clinical Genetics, University Medical Center Groningen, Groningen, The Netherlands and 17Department of Clinical Genetics, Erasmus Medical
Center, Rotterdam, The Netherlands
Email: Elisabeth MA Boormans* - [email protected]; Erwin Birnie - [email protected]; Hajo I Wildschut - [email protected];
Heleen G Schuring-Blom - [email protected]; Dick Oepkes - [email protected]; Carla AC van
Oppen - [email protected]; Jan G Nijhuis - [email protected]; Merryn VE Macville - [email protected];
Angelique JA Kooper - [email protected]; Karin Huijsdens - [email protected]; Mariëtte VJ Hoffer - [email protected];
Attie Go - [email protected]; Johan Creemers - [email protected]; Shama L Bhola - [email protected];
Katia M Bilardo - [email protected]; Ron Suijkerbuijk - [email protected];
Katelijne Bouman - [email protected]; Robert-Jan H Galjaard - [email protected]; Gouke J Bonsel - [email protected];
Jan MM van Lith - [email protected]
* Corresponding author
Published: 20 May 2008
BMC Pregnancy and Childbirth 2008, 8:18
doi:10.1186/1471-2393-8-18
Received: 21 April 2008
Accepted: 20 May 2008
This article is available from: http://www.biomedcentral.com/1471-2393/8/18
© 2008 Boormans et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: In the past 30 years karyotyping was the gold standard for prenatal diagnosis of
chromosomal aberrations in the fetus. Traditional karyotyping (TKT) has a high accuracy and
reliability. However, it is labor intensive, the results take 14–21 days, the costs are high and
unwanted findings such as abnormalities with unknown clinical relevance are not uncommon. These
disadvantages challenged the practice of karyotyping. Multiplex ligation-dependent probe
amplification (MLPA) is a new molecular genetic technique in prenatal diagnosis. Previous preclinical
evidence suggests equivalence of MLPA and traditional karyotyping (TKT) regarding test
performance.
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http://www.biomedcentral.com/1471-2393/8/18
Methods/Design: The proposed study is a multicentre diagnostic substitute study among
pregnant women, who choose to have amniocentesis for the indication advanced maternal age and/
or increased risk following prenatal screening test. In all subjects, both MLPA and karyotyping will
be performed on the amniotic fluid sample. The primary outcome is diagnostic accuracy. Secondary
outcomes will be maternal quality of life, women's preferences and costs. Analysis will be intention
to treat and per protocol analysis. Quality of life analysis will be carried out within the study
population. The study aims to include 4500 women.
Discussion: The study results are expected to help decide whether MLPA can replace traditional
karyotyping for 'low-risk' pregnancies in terms of diagnostic accuracy, quality of life and women's
preferences. This will be the first clinical study to report on all relevant aspects of the potential
replacement.
Trial Registration: The protocol is registered in the clinical trial register number
ISRCTN47252164
Background
In the Netherlands, invasive prenatal diagnosis is offered
to all women who are considered to be at increased risk
for Down's syndrome. Until recently, the vast majority of
invasive tests was done for advanced maternal age. More
recently, a nationwide screening program for Down's syndrome using first trimester serum testing combined with
ultrasonographic nuchal translucency measurement was
introduced [1,2]. A positive result of this combination test
or maternal age (36 years or older) represent 80% of the
indications for the invasive diagnostic procedures (amniocentesis and chorionic villus sampling). We have seen a
still continuing shift of maternal age-based karyotyping
towards prenatal screening based testing.
Traditional karyotyping (TKT) is considered the gold
standard for invasive prenatal diagnosis (PND) [2,3]. TKT
is able to detect a range of numerical and structural chromosomal abnormalities with considerable accuracy
(99.4–99.8%) and reliability[3,4]. However, TKT also has
several disadvantages: it is labour intensive and the costs
are high. Furthermore, obtaining results from karyotyping
takes 2–3 weeks, and this waiting time places a significant
emotional burden on women and their partners[5]. Moreover, the extensive detection capacity of TKT can be perceived as a disadvantage due to the detection of
abnormalities with unclear or mild clinical relevance.
These results can cause patient anxiety, emotional dilemmas concerning the continuation of pregnancy and, albeit
rare, unnecessary pregnancy terminations [6].
Due to these disadvantages, TKT has been challenged as a
reference test. In 2002, a molecular PCR-based technique,
MLPA (multiplex ligation-dependent probe amplification) became available to detect foetal aneuploidies in
amniotic fluid cells. In preclinical laboratory studies,
MLPA has been a robust test in detecting the most common chromosomal aneuploidies, namely trisomy 21, 13,
18 and sex chromosome abnormalities [7-9]. Compared
to TKT, MLPA has several potential advantages: the result
is available in 2–4 days instead of 3 weeks, the technique
requires only 2 ml of amniotic fluid instead of 16–20 ml,
and the technique is considerably less labour-intensive
and is suitable for high-throughput testing [8-10].
In the Netherlands, like other western countries, there is
an ongoing debate whether rapid molecular tests should
be used as a stand-alone diagnostic tool instead of karyotyping in prenatal diagnosis for certain indications[5,1115]. To strengthen the debate and to supply it with evidence, we designed a clinical prospective cohort study in
which MLPA is compared independently to TKT for the
two main referral indications; advanced maternal age and
increased risk following prenatal screening tests. The aim
is to estimate diagnostic accuracy for the detection of trisomies 21, 18,13 and sex chromosome abnormalities, as
well as the reduction in waiting time for the prospective
parents, maternal quality of life, women's preferences, the
relevance of 'unexpected findings' and costs.
Methods/Design
Aims
The M.A.K.E. study investigates whether MLPA can replace
TKT in prenatal diagnosis in terms of diagnostic accuracy,
costs and maternal quality of life for women undergoing
amniocentesis for the indications advanced maternal age
and increased risk following prenatal screening.
We hypothesize that MLPA has equivalent diagnostic
accuracy in detecting trisomies 21, 18, 13 and sex chromosome abnormalities. The reduction in waiting time for the
patient, maternal quality of life, women's preferences, the
relevance of 'unexpected findings' and costs are part of the
study.
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Study design
The M.A.K.E. study is a prospective, nationwide multicenter study. All eight genetic centres in the Netherlands
will participate.
Participants
Participants are pregnant women choosing to have amniocentesis for either advanced maternal age or increased
risk following prenatal screening.
Women undergoing amniocentesis for ultrasound abnormalities, parental chromosomal abnormality, or a previous child with chromosomal abnormality are excluded.
Procedures, recruitment and collection of baseline data
Pregnant women who visit one of the participating antenatal care units are informed by an obstetrician, midwife
or genetic counsellor on the different test strategies (prenatal screening and prenatal diagnosis) with associated
risks and benefits of each procedure. After they have chosen to have amniocentesis, eligible women will receive
participation information. After written consent, amniotic
fluid is obtained and tested with both MLPA and karyotyping. Stratification will be applied for each centre. Baseline demographic characteristics and obstetric history are
recorded. For the quality of life study and women's preference study a representative sub sample is drawn, with separate consent procedure.
Diagnostic tests
Details on amniocentesis are recorded in the case record
form, accessible through a secure website [16]. All amniotic fluid samples are tested with both MLPA and TKT.
Test results are blindly assessed. We chose not to fully
standardize the MLPA and TKT protocols but to accept the
minor differences within the eight laboratories in sample
preparation, formulation of the trisomy multiplex, equipment and interpretation of results. This will make the trial
more pragmatic, with better generalizability of the results.
The existing differences will be administered and will be
helpful in choosing the best test strategy in the nearby
future.
MLPA
In this study we use a commercially available kit, SALSA
MLPA P095 (MRC Holland, the Netherlands). For each
specific genomic target, a set of 2 probes is designed to
hybridize immediately adjacent to each other on the same
target strand. Both probes consist of a short target specific
sequence and a universal PCR primer-binding site. One of
the probes contains a so called 'stuffer sequence'. For each
probe in the multiplex, the stuffer part has a specific
length and sequence. After overnight hybridization to the
target DNA, each pair of adjacent probes is joined by a
ligation reaction. Next, PCR is performed with a single flu-
http://www.biomedcentral.com/1471-2393/8/18
orescent-labelled primer pair, which ensures that the relative yield of each of the PCR products is proportional to
the amount of each of the target sequences. The different
length products are separated on an automated capillary
sequencer and the peak areas are quantified[9].
Compared to other techniques available for rapid aneuploidy detection (RAD), i.e. quantitative fluorescence
polymerase chain reaction (QF-PCR) and fluorescence in
situ hybridization (FISH), MLPA has the advantage of
detecting 40 different loci in a single reaction. Furthermore, MLPA is suitable to tailor-made strategies for different clinical settings. Compared to FISH, MLPA is suitable
for high-throughput testing and less expensive. Compared
to QF-PCR, MLPA avoids the problem of non-informativeness of the polymorphic markers that may occur with
QF-PCR [17]. However, in contrast to QF-PCR, MLPA will
not diagnose 69, XXX triploidy.
Karyotyping
Karyotyping is a reliable and accurate test for detecting
chromosomal aberrations. The karyotyping detection rate
for Down syndrome is 99,5% [3].
The time from amniocentesis to diagnosis is 2–3 weeks, as
the test is non-automated and samples need to be cultured
in order to obtain foetal cells at the metaphase stage. Laboratory personnel involved in TKT are blinded for MLPA
results. In case of an abnormal MLPA result and a more
rapid test result is wanted, the earliest possible harvesting
of cell cultures is carried out.
Outcome measures
Primary outcome is diagnostic accuracy. Sensitivity, specificity and failure rates of MLPA are calculated for
(an)euploidies 21, 13, 18, X and Y and compared with
TKT as a reference test. Turnaround time on laboratory
and patient level is recorded. Discordant test results and
technical difficulties are counted, allowing for general and
per centre analysis. The effect of a learning curve if present
will be established.
Secondary outcomes
Quality of life questionnaires are filled out before amniocentesis and 2, 5, 14, 23 and 63 days after amniocentesis.
The questionnaires contain Spielberger's State Trait and
Anxiety Inventory (STAI), Impact of Event Scale (IES), Personal Perceived Control (PPC) and Medical Outcome
Study Short Form (SF-36) and questions on background
characteristics. We will compare two groups, one group,
receiving the result of TKT and the other group, receiving
results of both MLPA and TKT.
A discrete choice experiment will give insight in patient's
preferences and investigates the value pregnant women
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BMC Pregnancy and Childbirth 2008, 8:18
place on different characteristics of prenatal testing (e.g.
detection capacity, waiting time, consequences for the
child of the detected abnormalities).
The process of care is distinguished into three cost categories; direct medical costs (all costs in the health care sector), direct non-medical costs (costs outside the health
care sector that are affected by health status or health care)
and indirect costs of the pregnant women and her partner.
For each cost category, costs are measured as the volumes
of resources used multiplied with appropriate valuations
(cost-per-unit estimates, fees, national reference prices).
Statistical issues
Sample size
Adopting the results of karyotyping as the gold standard
and assuming that MLPA and karyotyping do not produce
different test results, that not more than 1/500 missed
cases of Down syndrome are accepted, and that the proportion of discordant test results is 0.002, we need at least
4500 paired test results to test the null-hypothesis of clinical equivalence (one-sided alpha 0.05, power = 1-beta =
0.90).
Data analysis
Data will be analyzed according to both the intention to
treat and per protocol analysis. To determine non-inferiority in diagnostic accuracy we will calculate absolute and
relative sensitivity, specificity and failure numbers of
MLPA and compare these to karyotyping (gold standard).
Furthermore, the time cytogeneticists or molecular geneticists need to provide an authorized test results and the
time needed to inform the patients is recorded. With these
data exact waiting periods for both tests can be estimated.
Quality of life outcomes. Repeated measurement analysis
will be performed for the between group evaluation.
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Discussion
MLPA can potentially replace the traditional karyotype for
detecting the most common chromosomal aneuploidies.
This study is designed to provide evidence on clinical
equivalency of MLPA compared to TKT in prenatal diagnosis. To determine the best test strategy in prenatal clinical practice for women undergoing amniocentesis for
either advanced age or increased risk following prenatal
screening, we will investigate diagnostic accuracy, speed,
maternal quality of life, women's preferences and costs.
Abbreviations
MLPA: Multiplex Ligation-dependent Probe Amplification, TKT: Traditional karyotyping, PND: Prenatal diagnosis, RAD: Rapid Aneuploidy Detection, QF-PCR:
Quantitative Fluorescent Polymerase Chain Reaction,
FISH: Fluorescent In Situ Hybridization
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
EMAB, EB, GJB and JMMvL were involved in conception
and design of the study. EMAB, EB and JMMvL drafted the
manuscript. All authors mentioned in the manuscript are
members of the M.A.K.E. study group. They participated
in the design of the study during several meetings and are
local investigators at the participating centres. All authors
edited the manuscript and read and approved the final
manuscript.
Acknowledgements
This study is funded by Zon-MW grant (945-27-045).
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